Retrovirus Production

Retrovirus Production

The development of retroviral delivery systems for mammalian cells has greatly enhanced the ability of scientists to deliver genetic material to cells of diverse origin in order to study the role of a particular protein or network of proteins in a specific cell type or biological process. The goal of the retrovirus production unit is to assist Cancer Center members with the preparation of high-titer retrovirus (e.g. lentivirus). The centralization of such services provides economy of scale, high-throughput production, increased biosafety, and quality control in the virus preparation process. The Facility provides a variety of services including prepackaged vector preparations, custom vector packaging, and vector titrations.

* All prepackaged vector preparations are VSVg pseudotyped, non-replicating lentivirus and come as filtered, concentrated vector in PBS. Concentration is performed by ultracentrifugation. All vectors, except pLKO-based, are quantitatively titrated using HEK293T cells and percentage of transduced cells determined by flow cytometry. Note: pLKO.1 vectors do not have a fluorescent marker. Therefore, the titer of these vector stocks has been determined by a p24 assay.

** The Wistar Institute Vector production unit has purchased the above plasmids from Sigma-Aldrich and Open Biosystems and does not profit from the sale of lentivirus in any way. All lentiviral vectors produced by the Wistar Vector Production Unit and distributed to Cancer Center laboratories are for use in research only.

Concentration of vector by ultracentrifugation

The Retrovirus Production unit will concentrate (~1000X) vector preparations by ultracentrifugation. All concentrated vectors are provided in PBS as 5-10 ul aliquots.

Vector titration

Vector production unit determines titers of vectors using a quantitative titration protocol on HEK293T cells, if the vector contains a fluorescent marker. Please do NOT request titrations on other cell lines or using alternative methods.

7. Due to variability in packaging of different retroviral vectors, production of a minimum titer cannot be guaranteed. Whenever possible, newly produced vectors should be titrated.

8. It is the responsibility of the investigator to register the intended specific use of recombinant DAN technology (i.e. retrovirus use) with Wistar’s Institutional Biosafety Committee (IBC) as required by the most current NIH Guidelines for Research Involving Recombinant DNA Molecules. For a full description of requirements, please consult the NIH Guidelines.

9. Please acknowledge the Protein Expression Facility if viruses are used in the generation of publication data.